V. Tomchuk, V. Gryshchenko, V. Tsvilikhovskyi, J. Illek

ENTEROCYTES MEMBRANES OF THE SMALL

INTESTINE AT PATHOLOGY AND CONDITIONS

OF HIBERNATION

Київ-2018

2

УДК 636.09:616.34

Т 56

Автори:

Томчук В.А., доктор ветеринарних наук, професор, академік Академії наук вищої

освіти України Грищенко В.А., доктор ветеринарних наук, професор, лауреат Державної премії

України в галузі науки і техніки

Цвіліховський В.І., кандидат біологічних наук, доцент

Іллек Й., доктор ветеринарних наук, професор

Рецензенти: С. П. Весельський, доктор біологічних наук, старший науковий

співробітник (Київський національний університет імені Тараса Шевченка)

С. А. Ткачук, доктор ветеринарних наук, професор, академік академії

наук вищої освіти України (Національний університет біоресурсів і

природокористування України)

Рекомендована до друку Вченою радою Національного університету

біоресурсів і природокористування України

(протокол № 3 від 24 жовтня 2018 р.)

Томчук В.А..

Т 56 Мембрани ентероцитів тонкого кишечнику при патології та за умов

гіпобіозу / В.A. Томчук, В.А. Грищенко, В.І. Цвіліховський. – К.: НУБіП

України, 2018. – 170 с.

ISBN У монографії представлено результати досліджень особливостей обміну

cAMP, cGMP і простагландинів в ізольованому епітелії тонкого кишечнику

великої рогатої худоби залежно від віку та при ентеропатології, а також

описано закономірності змін ліпідної компоненти мембран ентероцитів за цієї

патології (спонтанної та експериментальної) й переведенні тварин у стан

штучного вуглекислотного гіпобіозу, що лежать в основі адаптаційної відповіді

організму за дії екопатогенного чинника на рівні клітинних мембран.

Для фахівців у галузі клінічної біохімії, клінічної діагностики, фізіології і

патофізіології, терапії, морфології та фармакології, практичних лікарів

ветеринарної медицини, а також для магістрантів, аспірантів і докторантів,

науковців НДІ, науково-педагогічних працівників вищих навчальних закладів

ветеринарного та біологічного профілю.

ISBN УДК 577.115+619:616.3:636.2

ББК 28.072

© В. А. Томчук, В. А. Грищенко,

В. І. Цвіліховський, Й. Іллек 2018

© НУБіП України, 2018Preface

3

Nowadays, in medicine, the key role of the violations of the structural

organization of cell membranes in the development of severe liver diseases,

cardiovascular and nervous systems, disorders of many functions of blood cells and

the like is proved. In practical veterinary medicine, reparative therapy for internal

non-contagious animal diseases is a new and relevant approach, since the restoration

of the structural and functional state of the affected cells and the associated metabolic

processes does not end, and three to five weeks after clinical recovery.

Membrane structures of cells of a living organism are exposed to numerous

pathological factors of the external and internal environment. The interaction of the

damaging agent with the surface of the cell plasmolemus triggers a cascade of

interrelated biochemical processes proceeding both on the membrane and inside the

cells.

The intensity of restoration of intracellular homeostasis in the course of

development of pathology essentially depends on the duration of action of the

pathogenic factor and the adaptive capabilities of the organism to a large extent

determined by the degree of damage to the cell membranes. The main structural and

functional components of the lipid bilayer of cells are known to be phospholipids. So,

the functioning of cellular membrane systems depends on the integrity of their

phospholipid structures. At the same time, the usefulness of metabolic processes in

cells and their violation in the development of pathology is determined by the

structural-functional state of membrane systems.

Pathogenic factors affect any body cells, is characterized by a number of

regularities and features of the resulting changes at the molecular level. The complex

conduct of not only biochemical blood tests, but also indicators characterizing the

structural and functional state of cell membranes with their respective lesions is

important from the point of view of the development of effective therapeutic remedial

therapies, will help solve numerous complex problems with functional insufficiency

of internal organs and the study of molecular mechanisms development of animal

diseases. In this book, we describe the characteristic structural and functional changes

4

in cell membranes in the development of enteropathology and in the transfer of

animals to the state of artificial carbon dioxide hypobiosis.

We express our sincere gratitude to all those who contributed to the conduct of

relevant scientific research and the analysis of a significant amount of biochemical

indicators, first of all, to the staff of the Department of Biochemistry and Physiology

of Animals named after Academician M. F. Guliy.

Sincerely, the authors.

5

CHAPTER I

PECULIARITIES OF EXCHANGE OF cAMP, cGMP AND

PROSTAGLANDIN IN THE ISOLATED ESTHELICS OF THE SMALL

INTESTINE OF LARGE CATTLE IN DEPENDENCE ON AGE AND IN

ENTEROPATHOLOGY

Cattle are distinguished among other animal and human species by the

anatomical and functional features of the digestive canal [1–3]. Recent recent studies

have established the characteristics of molecular organization [4] and enzyme activity

[5, 6] in the cell membrane of the epithelium of the small intestine of cattle.

It is known that the hydrolytic and transport function of the cell membrane of

the small intestine epithelium is regulated by the intracellular concentration of cyclic

nucleotides – cAMP [7–9], cGMP [7] and prostaglandins [10], with predominantly

E2 and F1α. But in view of the fact that the biochemical and physiological

foundations of the processes of absorption and secretion in the small intestine of

cattle, with the exception of several reports [11], are not well understood, the

question of the state of biochemical regulatory systems (cyclic nucleotides and

prostaglandins) in the epithelium of a thin intestines of cattle.

In addition, acute digestive disorders with signs of diarrhea are widespread in

newborn calves in the first days of life, accompanied by dehydration of the body [11–

13] and loss of electrolytes [14–16]. In this case, the mucosa of the intestine

undergoes a significant disturbance of metabolic activity [17–19]. As is known, many

diarrhea-provoking factors of non-infectious etiology [20, 21] or infectious nature –

cholera [8, 13, 20, 22], salmonellosis [23], pathogenic E. coli, dysentery [24] develop

with the participation of cyclic nucleotides. Distinguish diarrhea according to the type

of action of cholera toxin (cAMP-mediated secretion of H2O and electrolytes) or the

action of thermostable toxin E. coli (cGMP-mediated secretion). The role of

prostaglandins in the initiation of secretion in the small intestine is also known [25]

and changes in their level in the mucous membrane in the pathology of the digestive

canal. But I will not mention the absence of a final opinion on the etiology and

6

pathogenesis of alimentary diarrhea in newborn calves [10, 13], as well as a similar

pathology in newborn children [26–28], and biochemical mechanisms (involving

cyclic nucleotides and prostaglandins) of the emergence and development of

enteropathology. At the same time, newborn calves, as an object of research, can act

as a natural model for studying this pathology, searching for effective preventive and

medicinal products.

It should be noted that in most of the available studies, the exchange of cyclic

nucleotides and prostaglandins was carried out using biopsies, mucosal scrapings or

intestinal sections, that is, in a material containing epithelial and other tissues in

addition to the task of using homogeneous preparations of isolated epithelium.

Proceeding from the foregoing, the study of the exchange of cyclic nucleotides

and prostaglandins in the epithelium of the small intestine of cattle, including healthy

and dyspeptic neonatal calves, has theoretical, clinical and practical significance.

In this connection, in Chapter I of this work problems of the development of a

technique for isolating isolated small intestinal epithelial cells of cattle, determining

the level of cAMP, cGMP, and the activity of cyclic nucleotide exchange enzymes in

the epithelium of the small intestine of adult cattle, newborn healthy and sick with

enteropathology calves, as well as the study of the content of prostaglandins E2 and

F1α in the epithelium of the small intestine of similar groups of animals.

7

1.1 Biochemical and physiological basis of the processes of absorption and

secretion in the small intestine

The small intestine is one of the main organs of the digestive canal and

performs a number of different functions: metabolic, secretory, transport-evacuation,

depositing, hormonal, protective; which to varying degrees ensure the

implementation of the two leading processes - hydrolysis and absorption of nutrients

[29–36]. The variety of these functions is due to the uniqueness of the structure of the

intestinal mucosa. To date, the structural and functional organization of the mucous

membrane as a whole, and in particular the epithelium covering it, has been

sufficiently well studied and described both in norm [29, 30, 37], and in the

pathology of the digestive canal [11, 16, 18, 19, 38, 39]. Therefore, it is rational to

outline the main features of the structure of the small intestinal mucosa and its role in

the processes of hydrolysis and transport of nutrients.

1.1.1 Structural organization of the mucosa of the small intestine

The mucosa of the intestine (lamina mucosa intestinum) is a complex structural

and morphological formation consisting of an interconnected complex of blood and

lymphatic vessels, the nervous, connective and contractile tissues, bounded from the

side of the intestinal lumen by the epithelium, and on the other hand by the muscular

membrane [6, 30].

Three distinctly differentiated layers are distinguished in the mucosa:

1) epithelial - representing a layer of intestinal villi - outgrowth of the mucous

membrane, protruding into the intestinal cough;

2) the layer of the mucous membrane proper, with indentations in it - intestinal

crypts;

3) a thin layer of smooth muscle tissue - muscle plate [6, 30].

The epithelial layer and its own layer of the mucosa are formed by a loose

connective tissue and are covered with a single-layered cylindrical epithelium.

8

Intestinal crypts are also lined with a single-layered epithelium, but its cells differ

substantially in structural, cytochemical and functional parameters from the villous

epithelium [6, 30]. According to the data obtained on the intestine of the rat, the

epithelial layer occupies 73%, the layer of the mucous membrane proper is 22%, and

the muscular shell – 5% of the volume of the intestinal mucosa.

The epithelial layer of the intestinal mucosa is the main component that

provides the realization of the processes of hydrolysis and transport of nutrients. It is

characterized by a folded surface in which the villous epithelium of the depression is

isolated. Between the villous epithelium and the epithelium of the indentation are

undifferentiated cells that have retained the ability to mitosis, from which the

proliferation of cell populations of villi and depressions occurs [6, 30]. The main

structural units of the epithelial layer of the intestinal mucosa are intestinal villi.

They are microorganisms with their vascular, muscular and nervous apparatus

[30]. Three types of villi are distinguished in form: leaf-shaped, finger-shaped and

linguiform [30]. At the base of the villi lies a layer of the mucous membrane proper,

with depressions around the villi – crypts. On each villus it is necessary up to 5~

9 crypts [6]. On the other hand, there are 10 to 40 villi per 1 mm² of the intestinal

mucosa, which increases the surface of the epithelium 8 times [32, 40]. In addition,

the surface area of the epithelium is increased by another 30–60 times due to a single-

layered cylindrical epithelium covering the intestinal villi. Its main mass (about 90%)

is made up of enterocytes with a narrow border formed by microvilli of the apical

plasma membrane [29, 30, 32]. The rest (about 10%) falls on other types of epithelial

cells [2]. Cellular elements of the mucosal layer proper are represented by reticular,

plasmatic and mast cells, lymphocytes, fibroblasts, acidophilic leukocytes and

macrophages [6]. In this layer, the presence of a significant amount of

mucopolysaccharides and fibroblasts is established, which indicates a large plastic

capacity of the mucosa [41].

The layer of smooth muscle tissue is represented by 2–5 crossing at an angle

layers of muscle cells covered with fibrous connective tissue [6].

9

The presented data on the general characteristic of the structure of the intestinal

mucosa testify to the significant importance of its constituent components in the

realization of bowel functions. However, since the main role in the processes of

hydrolysis and transport of nutrients belongs to the epithelial layer of the mucous

membrane, namely its functional unit – the cell of the intestinal epithelium, it is

necessary to dwell in more detail on its characteristics.

1.1.2 Functional-morphological characteristics of the epithelial cells of the

small intestine

The epithelium of the small intestine is characterized by polymorphism and

polyfunctionality even within one intestinal villi and one crypt [6]. It distinguishes

the following main types of epithelial cells:

1) intestinal epitheliocyte with a striated cicatrix, also called a cylindrical,

absorptive or main cell;

2) without bezel enterocyte intestinal crypt (undifferentiated, ancestral, stem,

maternal or cambial cell);

3) goblet enterocyte (mucoid cell or Goblet cell);

4) enterocyte with acidophilic granules (Packet cell);

5) intestinal argytafinocyte (enterochromaffinocyte, Kulchitsky cell or

endocrine cell) [15].

Intestinal epitheliocytes with striated edges have a cylindrical or columnar

shape with dimensions of 22–31 μm in height and 6–9 μm in width [39]. A distinctive

feature of these cells is the presence of two polar parts of the plasma membrane - the

apical and basal [29, 30, 37, 42]. The apical surface of the plasma membrane faces

the lumen of the intestine and is represented by a brush border, which consists of a set

of finger-shaped outgrowths of the cell membrane called microvilli [2, 29, 30, 36,

43].

The number of brush jaw micromirrors (from 1700 to 4000 on one cell) and

their sizes 0.8–1.5 microns in height and 0.05-0.1 microns in width vary

10

considerably, depending on the specific features of the intestinal cells, their level

differentiation, type of nutrition, age and functional state of the organism [2, 3, 6, 30].

Microvilli are a complex structure in which the overmembrane glycoprotein layer -

glycocalyx [2, 29, 30], the apical plasma membrane in the matrix is distinguished

[37]. The presence of a large number of hydrolytic and transport enzymes in these

structures ensures the unique function of the absorber cells of the intestine-the

hydrolysis of nutrients and their entry into the enterocyte [29, 31–37]. It is generally

accepted [31–36] that the digestive-transport complex of the apical plasma membrane

is the key link of the entire transport system of enterocytes.

The apical membrane of an enterocyte with a striated border into the basal one,

which faces the serous membrane of the intestine. It includes lateral areas, with which

the enterocyte borders on neighboring cells, forming intercellular slits and, in fact, the

basal part adjacent to the basal subepithelial membrane [6, 33, 42]. On the basal

surface of the plasma membrane, various enzyme systems that transport the

substances are detected mainly due to the energy liberated during the hydrolysis of

ATP (transport ATPases) [17, 44]. These enzyme systems transport and exchange

nutrients between the digestive system and the internal environment of the organism

[5, 6]. In addition, the basal part of the plasma membrane has an important role in

providing cell adhesion to the basal subepithelial layer [30]. Intensive carbohydrates

and lipid metabolism in absorptive cells of the intestine cause high activity in them of

biotransformation and detoxification systems [30]. These processes are provided by

the active functioning of enzyme systems localized in well-developed intracellular

organelles of enterocytes: mitochondria, the granular endoplasmic reticulum and the

Golgi complex. With regard to lysosomes, there are relatively few of them in

enterocytes and their main function is to release cells from the decay products of

intracellular structures at the final stages of their life cycle, and in the early postnatal

period to intracellular digestion [6, 30, 45].

Without bezel enterocytes are cells of intestinal crypts, from which mature

absorbent epitheliocytes and goblet enterocytes develop [6]. They are predominantly

cylindrical in shape and characterized by a weakly expressed striation, as well as a

11

few short and wide microvilli, which have a variable length and shape [46]. These

cells significantly differ from enterocytes with striated activity and localization of

enzymes in the plasma membrane. Thus, the activity of hydrolytic enzymes was

virtually absent in the rim of undifferentiated enterocytes [6]. However, these cells

have high proliferative activity, divide mitotically and replenish intestinal cell loss at

the tops of intestinal villi [6].

Goblet enterocytes are unicellular glands located both on intestinal villi and in

crypts [6]. The apical surface of these enterocytes is striated, as in absorbent cells. At

the same time, the number of microvilli on the plasma membrane of goblet

enterocytes is much smaller and they are of unequal length [47]. In addition, the

activity of enzymes in goblet enteronites is weaker than in the absorbent ones, and the

activity of hydrolytic enzymes has not been observed in their striated cortex [6]. The

function of goblet enterocytes is secretion into the lumen of the intestinal mucus,

which is rich in acidic and neutral mucopolysaccharides and poor in protein [6].

At the bottom of intestinal crypts are located enterocytes with acidophilic

granules (Panet cells). They have the shape of a truncated cone, wider at the base and

tapered to the apex [22]. Microvilli of these cells are rudimentary, very rare and

contain fibrils that penetrate not a few microns into the apical cytoplasm. Filling the

entire cytoplasm of Panet cells with large (from 2 to 4 microns) secretory granules

with a homogeneous material indicates their secretory properties. However, the

functional role of these cells has not been fully established [6].

Intestinal argytafinocytes (Kulchitsky cells) are triangular endocrine cells that

occur both among the epithelial cells of the intestinal crypts and among the

epithelium of the intestinal villi [6]. There are different variants of these cells. It is

established that some of them secrete serotonin, other cholecystokinin. However, the

functional role of intestinal argentafinocytes remains unclear [6]. So, the data

presented above show that all types of intestinal epithelial cells participate to a

greater or lesser extent in the implementation of complex processes of digestion,

absorption and transport of nutrients. The determining role in this undoubtedly is

assigned to the absorptive cells of the intestine - enterocytes with striated margins.

12

Mechanisms of absorption (absorption) and transport of substances in the

intestine include two interrelated processes - absorption and secretion. One of the

main components of these processes is the absorption and secretion of water and

electrolytes. The state of absorption in secretion within physiological limits is of vital

importance to the body. At the same time, abnormalities occur in the pathological

conditions of the digestive canal (diarrhea), which lead to a change in the

permeability of the intestinal epithelium to water and electrolytes. By this, it will be

logical to briefly dwell on the characteristics of the absorption and secretion

processes in the small intestine in norm and in pathology.

1.1.3 Characteristics of the absorption and secretion processes in the small

intestine in normal and pathological conditions

The digestive canal is a flow system in which there is an intense absorption of

water and salts, mainly sodium chloride [2, 6]. Of all the liquid and salts entering the

intestine, only 20% comes with food. The rest is formed as a result of endogenous

secretion of digestive organs, including the intestine [2, 30, 48]. Thus, it has been

established that water passes through the intestine of the cow 3–4 times, sodium in 6-

7 times, and chlorine 8-10 times more than in the case of a scorm [6].

The overwhelming amount of water and electrolytes is absorbed and secreted

in the small intestine [2].

Absorption and absorption of water is carried out by steady osmotic gradients.

In this case, water enters the body through conjugated active transport with

electrolytes, monosaccharides, amino acids, and also di- and tripeptides. The driving

force behind these processes is active sodium transport.

The absorption of sodium and chlorine ions is a conjugate process and from the

side of the apical membrane of the absorbing cells is realized by the electroneutral

mechanism, in which the absorption of sodium is carried out together with chlorine.

The sodium input is inhibited by the absence of chlorine in the gut lumen and vice

versa. This mechanism also includes anti-port systems: Nа+/К

+ , Cl

-/НСO

3-, Cl

- /OH

-,

13

and also Сl-/H

+ [49]. In addition, the absorption of sodium from the side of the apical

membrane is carried out by an electrogenic mechanism, also called joint sodium

transport with organic substances - glucose, amino acids. The driving forces of these

processes are transport enzyme systems (in particular Nа+, К

+-АТР-ase) located on

the basolateral membrane of the enterocyte and carrying out active transport due to

the energy released during ATP hydrolysis [17].

In crypt cells that secrete chlorine ions, the transport process also depends on

the electrochemical gradient that is produced by Nа+, К

+-АТР-ase of basolateral

membranes. However, in crypts, the carrier protein, sensitive to the sodium gradient,

is located in the basolateral membrane of the epithelium, and not in the apical

membrane, as in the absorptive cell. This contributes to the accumulation of chlorine

in the cell, which under normal conditions exits through the apical membrane along

an electrochemical gradient.

The general mechanisms of transport of water and sodium chloride described

above characterize the physiological state of the organism, in which the level of

absorption exceeds the level of secretion. However, the pathological state of the

digestive system (enteropathology with diarrhea phenomena) leads to serious

disruptions in the absorption of secretion, which in a short period of time lead to

dehydration [12, 15, 16], an acid-base balance, especially sodium [16]. Infectious

diseases such as cholera, dysentery, salmonella infection, pathogenic E. coli, as well

as pathologies of non-infectious nature-alimentary diarrhea, both in humans [20] and

in animals-cattle [11–13]. It should be noted that the biochemical mechanisms of

diarrhea ofinfectious nature have been sufficiently described to date, while the

widespread diarrhea of newborn calves [11, 13] and the similar problem in newborn

children have not been studied, which makes it difficult to conduct effective

preventive and curative activities in veterinary medicine and pediatrics.

14

1.1.4 Intracellular systems for the regulation of absorption and secretion

in the small intestine

Regulation of absorption and secretion in the small intestine is performed at the

level of the plasma membrane of the enterocyte by special intracellular mediators, the

main ones of which are cyclic nucleotides – cyclic adenosine monophosphate

(cAMP) and cyclic guanosine monophosphate (cGMP), as well as intracellular free

calcium [9, 50]. A unique feature of the epithelium of the small intestine is that all

these intracellular mediators have an effect on the ion transport processes by a similar

mechanism. Thus, an increase in the intracellular concentration of any of these

mediators inhibits absorption and stimulates the secretion of ions in the small

intestine of mammals.

The metabolites of polyunsaturated higher fatty acids, in particular

prostaglandins, are also important and intracellular regulators of the transport of

substances in the intestine [10].

In the context of the problem we are considering-digestive disorders in

newborn calves-cyclic nucleotides (cAMP and cGMP), as well as prostaglandins E2

and F1α, whose activation by various humoral, microbial and pharmacological agents

lead to a state of diarrhea [22–24]. Therefore, it is advisable to consider the function

of these intracellular regulators in more detail.

1.1.4.1 The role of cAMP and cGMP in the regulation of absorption and

secretion in the small intestine

Cyclic 3,5-adenosine monophosphate occupies a central place in intracellular

regulatory mechanisms, taking part in the implementation of a variety of vital

processes of the body; it controls cellular growth and differentiation, increases the

permeability of cell membranes, mediates the action of many hormones, participates

in the development of adaptive reactions and in the intracellular metabolism of

carbohydrates, proteins, lipids and mineral salts, as well as in neuromuscular

15

transmission and in the functions of the central nervous system [9, 50]. Cyclic 3,5-

adenosine monophosphate is formed in cells from ATP in the presence of Мg2+

ions

as a result of activation of the enzyme adenylate cyclase. In intestinal cells, this

enzyme is localized primarily on the basolateral membrane. Activation of adenylate

cyclase is carried out only by peptide hormones and neurotransmitters, as well as

toxins that do not penetrate the interior of the cell and bind to specific surface

receptors [9]. Therefore, adenylate cyclase is an entire system for signaling,

consisting of a hormone binding receptor, a catalytic component converting ATP to

cyclic AMP, and a complex of transducer proteins that bind the receptor and catalytic

components of the system. At the same time, the level of cAMP in the cell is

controlled by another enzyme, various forms of phosphodiesterase that are localized

both in the cytosolic fraction and in the membrane fraction and convert cAMP into an

inactive metabolite, a noncyclic 5'-adenosine monophosphate [50].

The mechanism of action of cyclic AMP is due to the activation of cAMP-

dependent protein kinases, which act as regulators of the corresponding metabolic

pathways [51]. The mechanism of protein kinase activation is the binding of cAMP to

the regulatory subunits of the enzyme, which leads to the release of catalytic subunits

that use ATP to phosphorylate the corresponding substrates.

Cyclic 3,5-guanosine monophosphate has similar cAMP functions. In the

intestine cGMP is formed in the brush border of enterocytes with the participation of

the enzyme guanylate cyclase and activates tissue-specific isoenzymes with GMP-

dependent protein kinase. The level of cGMP in the cell is also controlled by one of

the forms of phosphodiesterase, which cleaves with GMP to an inactive metabolite, a

non-cyclic 5'-guanosine monophosphate.

In the intestine, both cAMP and cGMP stimulate secretion. However, the

strength of the action, these mediators have some differences. Thus, it has been

established that cAMP and cGMP rabbits in the absorptive cells of the ileum inhibit

cotransport of Na and Cl equally, but in crypt cells the effect of cAMP is more

effective than cGMP. This can be explained by a decrease in the activity gradient

dependent on cGMP protein kinase from the villi region to the crypt region.

16

Thus, the data presented above demonstrate the important role of cAMP and

cGMP regulation of absorption and secretion in the intestine. At the same time, the

formation of cyclic nucleotides themselves can be controlled by other specific

substances called prostaglandins [50, 52], which perform a variety of functions,

associated with regulatory processes at the cell level.

1.1.4.2 The role of prostaglandins in the regulation of absorption and

secretion in the small intestine

Prostaglandins are biologically active substances that are cyclic hydroxy acids,

which are derivatives of polyunsaturated fatty acids with 20 carbon atoms [10, 53].

The main and most important precursor of prostaglandins is arachidonic acid. The

source of arachidonic acid is mainly the phospholipids of cell membranes [10]. The

release of arachidonic acid from the phospholipid pool of cell membranes is carried

out by phospholipases, in particular phospholipase A2. Further formation of

prostaglandins from arachidonic acid is accomplished by the operation of a special

enzyme called prostaglandin synthetase [10]. Prostaglandins are synthesized by

virtually all tissues of the body, but their number in different tissues and even in

different populations of cells of the same tissue are different. Synthesis of

prostaglandins occurs directly at the time of biological affect, and the action itself is

limited mainly to the place of their formation, since prostaglandins have a short half-

life and do not accumulate in the body [53]

Prostaglandins perform important functions, having different physiological and

pharmacological effects: they cause stimulation or relaxation of the cell, cause strong

diuretic and natriuretic effect, affect the tone of blood vessels, bronchi, blood

pressure level, cardiac activity, coronary blood flow, platelet aggregation, central and

autonomic activity nervous systems, hormone secretion and modulation of their

action in target tissues [10].

In the gastrointestinal tract there are mainly prostaglandins of the group E and

P, with a predominance in quantitative terms and in terms of the biological activity of

17

the prostaglandins of group E [53]. In addition to these prostaglandins, prostaglandin

A, D2 and prostacyclin have also been found in the intestine [53]. The biological role

of prostaglandins in the gastrointestinal tract is extremely diverse and consists in the

modulation of gastric acid and alkaline secretion, motor activity of the

gastrointestinal tract, the protection of cells vasodilation, and the role of mediators in

the inflammatory response in pathological conditions, as well as in local regulation of

electrolyte transport in the intestine.

Prostaglandins of both group E and group F stimulate active secretion of

electrolytes in the small intestine [53, 54], and the group F prostaglandin also

regulates intestinal motility [54].

Thus, it is obvious that the physiological level of cyclic nucleotides (cAMP

and cGMP) and prostaglandins in the epithelium of the small intestine plays an

important role in regulating the processes of absorption and secretion. At the same

time, as will be discussed below, a change in the concentration of these intracellular

mediators can lead to significant disturbances in biochemical processes in the small

intestine.

1.1.5 Biochemical basis for the development of acute digestive disorders in

the small intestine

Acute digestive disorders in the small intestine with the phenomena of diarrhea

are one of the most common pathologies of this organ. As a result of the development

of the disease, the processes of secretion over absorption take place. Therefore, the

severity of the disease, and often a fatal outcome, does not come from the direct

factor that causes diarrhea, but as a result of secondary processes - dehydration and

loss of electrolytes.

Factors that can cause diarrhea include: infectious diseases – cholera [8, 13, 20,

22], salmonellosis [23], E. coli, dysentery [24], viral; noninfectious – eating disorder

[20], diabetic diarrhea [55], lactose intolerance [21], caused by antibiotics or

laxatives. The existence of diarrhea of a neurohumoral nature is also described in

18

terms of the ability of certain hormones (vasoactive intestinal peptide, secretin,

serotonin) to cause a state of diarrhea.

It should be noted that the mechanism of action of many of the listed

pathological factors is now sufficiently well understood. By the type of effect

realization, they can be classified into several groups: acting by increasing the

intracellular concentration

1) cAMP;

2) withGMP;

3) acting through the system of Ca-calmodulin.

According to the cAMP-mediated pathway leading to diarrhea, cholera

diarrhea has been studied and described at the biochemical and pathophysiological

level, and diarrhea caused by thermostable toxin E. coli has been studied in the

cGMP-mediated pathway.

The current concept of the effect on the intestine of cholera toxin is presented

in the following form [8, 9].

The colonization of the small intestine by the bacterium of the cholera vibrio

(Vibrio cholere) leads to the accumulation of protein nature in the lumen of the

intestine. Cholera toxin consists of several subunits. The enzymatic subunit is in a

complex with subunits that bind to the apical membrane through the ganglioside site.

Subsequently, the subunit A is activated, giving peptide A1, which modulates the

adenylate cyclase of the basolateral membranes [8] in the following way. Adenylate

cyclase is a complex receptor-regulated enzyme complex, which consists of: 1 – a

membrane receptor; 2 – GTP-binding protein, which binds and hydrolyses GTP; 3 –

catalytic subunit of adenylate cyclase proper. Of these components, only the receptor

is localized on the outer surface of the cell [9].

Cholera toxin subunit A1 is an ADP-reabolizing enzyme that catalyzes the

addition of ADP-ribose (from the NAD+) to a GTP-binding protein, after which he is

not able to inactivate adenylate cyclase.

19

The result of exposure to cholera toxin is a prolonged activation of the

adonylate cyclase complex, which leads to a significant increase in the intracellular

concentration of cAMP [8, 9].

cAMP activates protein kinases, and those phosphorylate intracellular and

membrane substrates related to suction systems, modifications of passive

permeability and active transport.

It is assumed that these membrane substrates responsible for the inhibition of

absorption of Nа+ and Сl

- in the epithelium of the villi and the activation of C1 ~

secretion in the epithelium of the crypts.

E. coli thermostable toxin has a similar structure to the cholera toxin and

probably likewise stimulates guanylate cyclase activity [56] is localized in the apical

membrane. At the same time, the concentration of cGMP increases, resulting in a

secretion of Н20 and electrolytes in the intestinal cavity.

At the same time, there are a number of factors that distinguish the cGMP-

mediated mechanism of diarrhea from the mechanism involving cAMP.

First, the thermostable toxin of E. coli does not bind to membrane

gangliosides. Secondly, the modulation of the secretion processes in the intestinal

epithelium is carried out by mutually independent targets for cAMP and cGMP,

presumably by the domains of the corresponding protein kinases. Third, in the

presence of E. coli heat-stable toxin saturation plots for cGMP increases 5 times for

3–5 minutes incubation, cAMP and only 2 times within 2 pm. In addition, the

thermostable toxin of E. coli has a more complex mechanism of action. It has been

found that the thermostable toxin similar with E. coli diarrhea has the ability to

induce the calcium ionophore A-23187, which is known to promote entry of Са2+

into

the cells by concentration gradient. Moreover, A-23I87 intensified the action of toxin,

activated the activity of calmodulin and phosphodiesterase. The blocker of Ca-

channels, verapamil, and the calmodulin inhibitor-trifluoropyrazine, removed the

ionophore effect. The authors concluded that the increased secretion of Nа+ and Сl

-

diarrhea stimulated thermostable toxin of E. coli, it may be associated with an

20

increased flow of Са2+

and calmodulin activity in the microvilli of the intestinal

epithelium.

It should be noted that the Ca-dependent mechanism has a significant place in

the characterization of secretory diarrhea. This should include the already mentioned

factors – A-23I87, as well as serotonin, neurotensin, carbachol, ricinoleic acid,

deoxycholate. Being an activator of phosphodiesterase and an adenylate cyclase

inhibitor [8], calcium ions together with calmodulin are able, without changing the

level of cAMP and cGMP, to stimulate intestinal secretion by activating protein

kinases. At the same time, it is assumed that the primary effect of cAMP and cGMP

can also be manifested through an increase in intracellular Са2+

.

Prostaglandins activate adenylate cyclase, increasing the intracellular content

of cAMP, and are also capable of causing diarrhea. Intraperitoneal injection of

prostaglandin E2 (4 μg/kg body weight) causes diarrhea as early as 15 min, whereas

prostaglandin F2α caused a similar effect only after 30 min. The content of

prostaglandin E2 in blood plasma for diarrhea is 894 pg/cm³ at 346 pg/cm³ in healthy.

All this indicates that prostaglandin E2 relates to secretory diarrhea, then F2α refers

to bowel motility. At the same time, the involvement of eicosanoids in the

inflammatory processes and the presence of inflamed foci in the intestine with

diarrhea casts doubt on the concept of their main causal role in the development of

diarrheal syndrome.

Thus, regardless of the way in which the effect of the external factor is

realized-cAMP or with the GMP-mediated mechanism, Са2+

or Са2+

-calmodulin-

dependent pathway, the absorption processes are inhibited and/or H2O, Nа+, CI

-

secretion processes are stimulated apical and basolateral membranes.

As for other manifestations of acute digestive disorders of infectious origin -

salmonellosis [57], dysentery [58], they are also characterized by an increase in

cAMP in the epithelium, appear to follow the pathway described for cholera toxin.

Little studied is diarrhea of a neurohumoral nature and of an alimentary origin,

associated with malnutrition and widespread in newborns, both in humans [20, 28,

42] and in animals [11, 29]. This raises the problem, on the one hand, of deciphering

21

the biochemical mechanisms of this pathology occurring at the level of the intestinal

epithelium, and on the other hand, the search for effective preventive and therapeutic

agents. As noted above, the main functional load in the processes of absorption and

secretion is performed by the epithelial cell membrane - morphologically divided into

the apical and basolateral parts (membranes). Although the technique of human small

intestine biopsy has now developed sufficiently, the application of this procedure in

pediatrics is not acceptable. An alternative is modeling diarrhea in laboratory animals

or studying in vivo, using newborn calves as an object of study, in which the

enteropathology of non-infectious nature is widespread [11, 29].

As was established earlier by our studies [16, 17, 39], the molecular

organization of the apical and basolateral membranes of the epithelium of the small

intestine of cattle, with the exception of some differences, is similar to that of

monogastric animals. In the same studies, as well as by other authors, lipid

composition changes were established [16, 59] and transport ATPases [16, 17, 39] in

the cell membrane of the epithelium of the small intestine, which, apparently, is

typical for acute digestive disorders, since similar changes have been described for

selmonellosis infection [23, 59], or with the action of diphenyl laxatives. The study of

diarrhea in newborn calves, besides this, is of practical importance for animal

husbandry. Therefore, studies of the mechanisms of the development of

enteropathology with diarrheal syndrome in newborn calves, including the possibility

of participation in this process of cyclic nucleotide exchange systems – cAMP and

cGMP, prostaglandins, or other biochemical systems described in this review, have

important scientific, theoretical and practical implications.

In conclusion, it should be noted that the conclusions about the role of cAMP

and cGMP in adsorption and absorption processes in the small intestine are often

based on measuring the total content of these nucleotides in the mucosa including, in

addition to the epithelial mucosa, other tissues of the mucous membrane – muscle,

fibroblasts, blood, etc. Hence the difficulty in interpreting the results obtained – on

the contribution of a tissue to the content of cGMP or cAMP. Therefore, to

adequately study the possible role of cAMP, cGMP or prostaglandins in the processes

22

of absorption and secretion in the small intestine, it is necessary to use isolated

epithelium, excluding the admixture of cells of other tissues.

23

1.2 Obtaining isolated cells of small intestine epithelium of cattle

As is known, for the study of biochemical parameters of the small intestine

epithelium, the use of isolated epithelial cells is the most acceptable, which makes it

possible to exclude the effect on the studied parameters of the contribution of cells of

other tissues present in such complex formations as the intestinal mucosa,

To date, there are many methods developed for different experimental

conditions and different research objects. At the same time, there are no reports in the

literature of a method for obtaining cells from the small intestine of cattle. Therefore,

in this part of the paper, the results of studies on the selection of optimal experimental

conditions and the effective method of obtaining isolated cells of the small intestine

epithelium of cattle are described.

The research used cattle of black and motley breed. Before the research in the

educational establishment, experimental groups of animals were clinically healthy

adult cattle aged 3–5 years, newborn calves aged 3–5 days – healthy and sick with

acute digestive disorders.

Adult animals of cattle were used in experiments according to two schemes,

depending on the tasks of the experiment:

a) to develop a method for producing isolated epithelial cells, small intestine

sites were selected in a meat-packing plant, washed with 0.9% NaCI, pH 7.4 and

transported to the place of studies in the cold at 4–8 ° C;

b) studies of metabolism, the state of exchange of cyclic nucleotides, enzymes,

prostaglandins, were carried out in isolated epithelium obtained from experimental

animals, as described below.

1.2.1 Preparation of intestinal epithelial cells by various methodological

approaches

Originally, the chemical citrate-EDTA method was studied, as the most simple

and economical. Sequential incubation with solution (A) containing sodium citrate

24

for 10 min, and then solution (B) for 15 min, allows a suspension of single epithelial

cells from the mucous membrane that retain the morphological structure and polarity

(apical and basal parts) when analyzed by light microscopy. We have also studied the

possibility of using the enzymatic method of obtaining epithelial cells, since other

methods-scraping, vibration, rotation of the intestine [33] are not currently applied.

The enzymes chosen were the most widely used enzymes - hyaluronidase,

collagenase and the method developed [60] using the proteolytic complex Aspergillus

oruzae under the brand name Aсrizim-ІІІ ("Diagnosticum", Lviv).

Criteria for the effectiveness of the application of these enzymes were selected:

a) the ability of enzymes to maximal release into the incubation environment of

epithelial cells; b) the absence of large aggregates and epithelial beds; c) the

homogeneity of the suspension; d) the nature of the cells obtained; e) satisfactory

values of intravital metabolic parameters.

As a result, it has been established that Aсrizim-ІІІ possesses the minimum

efficiency in the separation of epithelial cells from 1 cm² of the intestinal wall (along

the serous membrane), and the maximum – of collagenase. Hyaluronidase reached

81% of the collagenase efficiency for 5 min, 77% for 15 min and 69% for 30 min

incubation, but at the same time, the presence of aggregates and epithelial layers was

greatest when using hyaluronidase and especially the citrate/EDTA method, as was

noted in [61]. In the case of the use of collagenase and, especially, Aсrizim-ІІІ, the

cell suspension was devoid of aggregates, was homogeneous, and after precipitation

and washing by centrifugation (in the angular rotor) with light shaking, it again

turned into a homogeneous suspension.

Thus, by the efficiency of cell separation, the homogeneity of their suspension,

the amount of material, the most appropriate is the use of collagenase. Most cells

retained a monologic polarity with a clearly distinguishable apical (Figure 1.1, 1.2)

(macroverse) and a basolateral particle, an elongated cylindrical shape with a brush

border on the top, structured by the cytoplasm.

Fig. 1.1. Electron microscopic, characteristic of isolated small intestine cells of cattle, obtained by various methods: a –

0.05 % collagenase; b – 0.1 % of hyaluronidase; c – citrate/EDTA

The arrows indicate the high native activity of the apical and basal part of the cell membrane (a), the damage to the apical

membrane (b), the presence of clusters of cells (c). x 7200.

Fig. 1.2. Electron microscopic characteristics of isolated small intestine

epithelial cells of cattle, obtained with 0.1% Aсrizim-ІІІ (a).

A typical epithelial cell: 1 – nucleus; 2 – the basolateral membrane; 3 – apical

membrane; 4 – mitochondria; 5 – area of apical intercellular contacts.

27

At the same time, the incubation of cells under physiological conditions for

120 min revealed a high yield of cytosolic enzyme lactate dehydrogenase from cells

obtained with the help of Aсrizim-ІІІ, which may indicate their low viability.

1.2.2 Evaluation of the metabolic activity of the epithelium obtained by

various methodological approaches

The characteristics of the metabolic and functional characteristics of the

isolated cells is crucial for the entire technique. This is especially important if it is

necessary to maintain a culture of epithelial cells. The criteria for evaluating cells

include a number of biochemical (lactate, pyruvate, citrate, etc.) and functional

(transport of amino acids, sugars, inclusion of nucleotide bases) indicators. In some

cases, the main goal is the speed of the study of the required indicators, and therefore

there is no need for a thorough evaluation of the preparations obtained.

In view of the fact that epithelial cells were used immediately in our studies,

the evaluation of their metabolic activity in obtaining enzymes differing was reduced

to the study of glycolysis substrates as the main way of energy supply to the intestinal

epithelium [62].

As might be expected, depending on the procedure, differences were found in

the content of pyruvate, lactate, in the kinetics and specific activity of lactate

dehydrogenase. It is known from the literature that the treatment of cells with

hyaluronidase is more severe [25] than the use of collagenase [60]. As shown by

studies using Akrizim-ІІІ [60] to isolate the pyramidal neurons of the rat

hypocampus, the amplitude of the chemoactivated currents in the neuron was an

order of magnitude higher (0.724 and 8.520 nA) compared to collagenase. The

advantages of Aсrizim-ІІІ before collagenase, in these studies, was confirmed by a

similar difference in the registration of potential-dependent sodium currents.

The effect of these enzymes on the epithelial tissue of the small intestine of

cattle has its own characteristics - the highest metabolic activity was detected by the

action of Akrizim-ІІІ, and the smallest - with the use of collagenase. Taking into

28

account the data of electron microscopic studies and low cell viability during the time

of incubation with the use of Akrizim-ІІІ, it can be concluded that the increase in

metabolic activity in this case is aimed at replenishment of energy costs, which in

turn are aimed at strengthening the life support systems of the cell (Nа+, К

+ pump).

In support of this view, similar collagenase effects of opposite direction are

directed: with a high yield of cells, high plasma membrane safety for 120 min after

the production of isolated epithelial cells, opposite metabolic activity is observed in

comparison with Aсrizim-ІІІ.

Thus, taking into account the indices of cell viability, the amount of material

obtained, we propose two working schemes for isolating isolated cells of the small

intestine epithelium of cattle using: a) 0.05% collagenase, as a highly specific method

of cell disaggregation; b) citrate/EDTA, as a sufficiently effective and, at the same

time, economical method.

1.2.3 Scheme of obtaining cells of the small intestine epithelium of cattle

The use of collagenase involves the following steps. After selection of the

intestine and removal of chyme, it is washed with physiological saline, pH 7.4, which

has been cooled to 4–6° C. A segment of the intestine 10–15 cm in length is turned

out and placed in an incubation medium (see mat. and meth.) at 37° C, which is pre-

blown to saturation (4–6 min) with 95% 0.2 and 5% С02, and before application of

the intestinal region, the collagenase enzyme is added to a concentration of 0.05%

and incubated for 15 min with a gentle shaking at a frequency of 45–60 times per

minute.

At the end of the incubation, the intestinal tract is removed and the cell

suspension is filtered through 4 layers with a pre-moistened gauze incubation

medium. The resulting filtrate is centrifuged at 500 g in an angular rotor for 5 min.

The supernatant is discarded, and the precipitate is diluted with an oxygenated

incubation medium but not containing collagenase and centrifugation is repeated

under the same conditions. The cell washing procedure is repeated 2–3 times. The

29

final precipitate of isolated epithelial cells is resuspended in isolation medium to a

concentration of 1–4 mg protein cells/cm³.

Thus, the developed procedures for obtaining isolated cells of small intestine

epithelium of cattle using collagenase and citrate/EDTA are quite acceptable for

studying various intravital biochemical parameters, comparative studies in the age

range or for studying pathologies of the digestive canal – enteropathology with

diarrhea phenomena.

1.2.4 Characteristics of energy metabolism in the epithelium of the small

intestine of adult cattle, healthy and sick enteropathology of newborn calves

Features of nutrition and digestion of cattle are now widely reflected in the

research of many scientists, which makes it possible to identify cattle for organizing

the digestive canal among other animals. Studies Melnichuk D. A. et al. [4, 5, 12, 16–

19, 30, 39, 50, 63–74] established the structural and functional characteristics of

subcellular elements of the small intestine epithelium. In this case, differences in the

lipid composition of the epithelial cell membrane [4, 70] of enzyme activity [16, 17,

39] were established in norm and in pathology in acute digestive disorders. To date,

there are a number of differences in biochemical parameters between newborns and

adults [4, 37, 68]. Taking into account that the hydrolytic, transport, regulatory

(cAMP and cGMP) function of the epithelium has a high dependence on energy

supply, in this part of the work the characteristic of the state of energy metabolism in

the epithelium of the small intestine of adult cattle, newborns healthy and sick with

enteropathology of calves is given.

For the content of metabolites of glycolysis and the cycle of tricarboxylic acids

in isolated small intestine epithelium of cattle, it is possible to characterize the degree

of metabolism in adult animals, especially in newborn calves and changes in

pathology. Thus, in adult cattle, a low level of pyruvate, characteristic for glycolysis,

can be noted with a high lactate content. The lactate/pyruvate ratio is 37. Newborn

healthy calves, compared with adults, significantly lower lactate levels, along with a

30

low content of pyruvate, citrate and lactate dehydrogenase activity. In newborn

animals with enteropathology, in comparison with healthy animals, the content is

increased: 3 times – pyruvate; 2.5 times lactate; in 18,8 times – citrate and 2,4 times

lower the activity of lactate dehydrogenase.

It is known that the main energy source of the epithelial tissue of the small

intestine is glycolysis. Despite the fact that the characterization of metabolic

processes in the intestinal epithelium is widely covered in the literature, interpretation

and comparative analysis of metabolic indicators are difficult due to the use of

various drugs – intestinal or mucosal scrapings, which contain cells of other tissues .

In this respect, the papers, where isolated epithelial cells were used, deserve attention.

According to the data of, the concentration of lactate in the small intestine epithelium

of the rat is 16 mM and, according to the authors, is 50 times higher than the pyruvate

content.

In original studies using proton NMR spectroscopy in the chicken epithelium,

the lactate level is 6.5 mM. If we take the intracellular volume of 1 mg of cells within

5–10 μl, then the level of lactate in the epithelium of bovine animals will be about

73–36.5 mM, according to our data. This can be explained either by the specific

feature of the research object – cattle, the main product of which is the carbohydrate

food [1–3, 26, 59], or reflect the typically high glycolysis level characteristic of this

small intestine-jejunal section [23].

At the same time, the specific activity of lactate dehydrogenase in bovine

epithelium is commensurate with the data obtained for isolated rat epithelium [47].

The low level of metabolites in healthy newborn calves, on the one hand, does

not agree with sufficiently intensive transport processes during this period of

development [29], but on the other hand it can be explained by the receipt of many

components of energy metabolism from colostrum or characteristic of newborns state

of carbohydrate metabolism. As shown [14], the rate of glucose oxidation in the

intestinal sections of the intestines of newborn rats is very low until 21 days of

development – during the period of feeding them with mother's milk. When

switching to independent feeding, the rate of glucose oxidation and lactate production

31

increases 3–4 times [14]. This explains the low level of lactate, citrate, pyruvate in

healthy newborn calves. The pathological state of the digestive canal leads to a

violation of the electrolyte balance of the organism [12, 14–16] and affects the

function of the epithelium [13, 18, 19, 24, 26, 38, 39, 41, 59], and according to our

data changes intracellular homeostasis. The high level of citrate (18.8 times higher) in

the epithelium of the small intestine of patients with enteropathology of newborn

calves, in comparison with healthy calves, indicates the inhibition of oxidative

processes in the mitochondria, which may be due either to insufficiency of the

oxygen supply of the epithelium [36], or to inhibition by another mechanism.

According to [30], suppression of oxidative phosphorylation by oligomycin or

rotenone leads to a change in the course of glycolysis activation.

Proceeding from this, the increase in the level of lactate observed in the

epithelium of newborn calves during diarrhea indicates activation of the glycolysis

processes. In addition, the condition of animals during the period of illness can be

conditionally regarded as a state of hunger, which can also lead to the activation of

glycolysis [40]. At the same time, a decrease in the specific activity of lactate

dehydrogovase in the epithelium in enteropathology can be explained by inhibition

by a high level of pyruvate. In addition to the presented results from the calculated

value for the NAD+/NADH ratio, which is 242.7, 547.7 and 659.2 respectively for

adult cattle, newborns healthy and diarrhea patients, the conclusion is that in the

epithelium of patients animals are inhibited by reductive reactions.

Thus, isolated small hamstring epithelial cells isolated with collagenase or

citrate/EDTA retain their metabolic parameters, native cell membrane, morphological

polarity and can be recommended for studying various biochemical characteristics.

Evaluation of the metabolic activity of the epithelium indicates differences in the

state of energy metabolism in healthy newborn calves and significant changes in

enteropathology. The use of isolated epithelial cells to study the state of exchange of

cyclic nucleotides and prostaglandins, according to the procedure proposed by us,

undoubtedly has a number of methodological advantages over other forms of drugs -

mucosal scraping, intestinal wall.

32

1.3 Characteristics of intracellular regulatory systems in the epithelium of

the small intestine of adult cattle, healthy and sick with enteropathology of

newborn calves

The epithelium of the small intestine performs a variety of various and

numerous functions associated with the processes of absorption and secretion of

nutrients, water and electrolytes. To ensure these functions, a number of regulatory

systems exist in the epithelium, including a classical scheme involving cyclic

nucleotides – cAMP and cGMP, calcium, calmodulin and possibly prostaglandins.

The functioning of these intracellular regulatory systems is based on maintaining

their concentration within the cell at a certain basal level, which depends on the

functional state of the organism, the intestinal tract and, apparently, the species of the

animal.

It should be noted that there are no reports in the literature that characterize the

state of exchange of cyclic nucleotides and prostaglandins in the epithelium of the

small intestine of cattle. In addition, the biochemical mechanisms for the

development of acute digestive disorders in newborn calves have not been fully

investigated, although they are of great importance for modern animal husbandry for

the purpose of preserving young animals, and may also be an alternative model for

studying similar pathologies in pediatrics.

1.3.1 Cyclic nucleotide exchange state

When studying the state of cyclic nucleotide exchange, we studied the content

of cAMP, cGMP and their exchange enzymes in freshly isolated isolated cells of the

small intestine epithelium of adult cattle, newborns healthy and sick with

enteropathology of calves. Given the likelihood that the gastrointestinal tract may be

a source of cyclic nucleotides in the blood, and the possibility of filtering the cyclic

nucleotides in the kidney [52, 74, 75], the content of cyclic nucleotides was also

studied in blood plasma and urine experimental animals.

33

The content of cAMP in the epithelium of adult cattle is 59.4±10.9 pmol/mg

protein. In healthy newborn calves, the value of this indicator was not significantly

different from adult animals and was 46.21±20.6. Comparison of the obtained indices

with analogous literature data is difficult, in view of the absence of studies using

isolated cells. Although, according to [76], in the mucous membrane of developing

rats, the level of cAMP is higher at a young age.

A high level of cAMP in the postnatal period is also characteristic of other

tissues and organs - the liver, skeletal and cardiac muscles of adipose tissue [60]. In

patients with diarrhea of calves, the level of cAMP decreases by 5.7 times. It is

known that with enteropathology accompanied by diarrhea, on the contrary, there is

an increase in the content of cAMP in the intestinal wall or mucosa in cholera [60,

75], salmonellosis [23], dysentery [74]. Thus, according to Bunin K.V. et al., the

level of cAMP in biopsies of the human small intestine rises from

2346±310 pmoles/g of crude tissue in healthy to 4,583±789 in patients with

dysentery. Similar changes from 817±23 to 1120±63 pmol/g of raw tissue were

detected by Sidakov B.M. et al. in dysentery or other authors [23] for salmonella

infection (from 335 to 1254 pmol/g crude tissue).

Along with this, it was shown that the level of cAMP in the intestine depends

on the consumption of milk [60]. Deprivation of 5-day rats of milk leads to a

significant decrease in the level of cAMP in their small intestine compared to the rats

of the same age who receive milk. Even digested milk can serve as a source of cAMP

in the intestinal mucosa. But these data are unlikely to be involved as the only

explanation for the low level of cAMP in the intestinal epithelium of diarrhea-sick

calves, who are often on a starvation diet and who are being reduced and sometimes

given away colostrum. It is known that consumption of solid food in the period after

weaning from milk increases the concentration of cAMP in the mucosa [60].

The content of cAMP in the blood plasma of adult cattle is more than 2 times

higher than that of human [74] and rat [60] and is possibly determined by the high

level of cAMP in the intestines of cattle at about 6,000 pmol/g wet weight cells, while

newborn calves it is close: 15.5±2.2 pmol/ml in newborn calves, and 13–14 pmol/ml

34

in humans [23, 24, 74]. According to the data of [74], the content of cAMP in human

plasma under dysentery did not differ between healthy and sick, and in the patients'

urine was slightly higher.

The content of cAMP in the blood plasma of healthy and sick newborn calves

is consistent with the indices in dysentery [74], whereas in the urine there are no

significant differences. Moreover, the level of cAMP in the urine of adult animals is

significantly (3 times) lower than in healthy newborn calves.

Comparison of the presented indices between groups of animals in the age

aspect and in pathology, as well as in the intestine - blood - urine chain is advisable to

conduct after considering the level of another cyclic nucleotide – cGMP and enzymes

of their metabolism.

Thus, the content of cGMP in isolated small intestine epithelium of adult cattle

and healthy newborn calves is in trace amounts, whereas in patients with

enteropathology of newborn calves it is 10 pmol/mg protein, i.e. an order of

magnitude higher than in healthy animals.

In the studies mentioned Bunin K.V. et al., when dysentery increased the

content of cAMP in the epithelium, there was no increase in cGMP, and even vice

versa, its decrease was observed. As we see, a similar but opposite direction in the

content of cAMP and cGMP was established by us in the enteropathology of newborn

calves.

In the blood plasma of adult cattle and healthy newborns, unlike humans (3–

9 pmol/ml), the content of cGMP has trace amounts, and only in patients with

enteropathology approaches 2.7±0.8.

At the same time, the results on the content of cGMP in the urine of cattle are

fully interpreted. The high content of cGMP in the urine of adult cattle

(10.3±2.1 nmol/ml) and healthy newborns (2.3±0.6 nmol/ml) is 20 times higher than

the content of cAMP in human urine. There are in view of the peculiarities of feeding

adult cattle (high water content in plant foods) and feeding newborn calves

(colostrum, milk), and there is a need for intensive cGMP-dependent glomerular fluid

filtration [7]. In newborn calves with diarrhea, intensive dehydration of the body

35

through the intestine occurs, and diuresis, in all probability, is inhibited, as is

observed in enteropathology of infectious origin [74]. Hence, the content of cGMP in

urine is 44 times lower than in adult cattle, and 10 times lower than in healthy

newborn calves.

Thus, the obtained results indicate that cattle, as polygastric and ruminant

animals, have a number of features in the content of intestinal epithelium, blood, and

urine, with respect to the content of cAMP and cGMP, which are reduced to an

increased cAMP content and a decreased cAMP level in blood plasma compared with

other animals and humans, a high level (44 times) with cGMP in the urine.

For diabetic patients of newborn calves, the content of cGMP in isolated small

intestinal epithelium increased by more than 10 times against the background of a

lower content of cAMP in it. Moreover, a low level of cAMP in the epithelium can

not be associated with a pool of adenyl nucleotides, as in the previous chapter it is

shown that in patients with enteropathology of calves the state of energy metabolism

(glycolysis) is in a sufficiently activated state.

Given the mechanisms of the development of enteropathology of infectious

and non-infectious etiology, we are inclined to discuss the results obtained in terms of

the mechanism of cGMP-mediated secretion in the small intestine in diarrhea of

newborn calves.

1.3.2 The activity of adenylate cyclase, guacylate cyclase and

phosphodiesterase

The activity of adenylate cyclase in isolated cattle small intestinal epithelial

cells has a slightly lower activity than that described for other tissues [7, 9, 66, 77–

79] and does not differ from that in healthy newborn calves. The established values of

the activity of adenylate cilase correlate with the content of cAMP in the epithelium

of these animals.

36

At the same time, in patients with enteropathology of newborn calves, the

activity of adenylate cyclase is reduced by 35% compared to healthy calves (cAMP

by 5.7 times).

It is known that the adenylate cyclase complex is sensitive to many regulatory

factors: hormones, neurotransmitters, calcium and calmodulin, the phospholipid

composition of membranes [78, 80–82], viscosity [52], the presence of an activating

substrate-GTP , and also have age features. Thus, the interpretation of data on the

change in the activity of adenylate cyclase in the epithelium of the small intestine of

patients with enteropathology of calves can be very versatile.

First, the factor initiating diarrhea (increasing the level of cGMP) is unknown,

since in the cAMP-mediated pathway of diarrhea development, the reverse is

observed-activation of adenylate cyclase [23, 24, 74].

In our previous studies [4, 17, 74], it was established that in the basolateral

membranes of the small intestine epithelium in diarrhea of calves there is a change in

the phospholipid, fatty acid composition and cholesterol content, ie, precisely in that

part of the cell membrane where adenylate cyclase is localized [32] . Changes in the

lipid composition can lead to a change in the viscosity of the membranes, which in

turn, can affect the activity of the enzyme [7, 9].

However, such an explanation is one-sided. It is likely that the factor that

induces the production of cGMP can simultaneously be an inhibitor of cAMP

production. Such phenomena include an increase in the concentration of intracellular

calcium and the activation of calmodulin [81–83], which are capable of activating

guanylate cyclase.

Indeed, the activity of guanylate cyclase in the epithelium of the small intestine

of patients with enteropathology of calves (14.3±0.61 pmol/mg protein per 1 minute)

is 2.2 times higher than in healthy calves. Based on these data and high-level data in

the cGMP epithelium, it can be concluded that the secretory processes in diarrhea of

newborn calves go along the cGMP-mediated pathway.

The enzyme, hydrolysing cyclic nucleotides – phophodiesterase, also has

features depending on age and enteropathology. Its specific activity in the epithelium

37

of cattle is close to the described activity in the homogenate of the mucosa of the

rabbit jejunum [23]. In newborn calves, the activity of phosphodiesterase is more

than 2 times lower than in adults, and in patients with enteropathology, on the

contrary, it is associated with such activity. The latter could be explained by previous

studies [4], where in the brush border and basolateral membranes of newborns, and

especially patients, the presence of myristic acid, an increase in the content of oleic

acid, which, as shown in the studies of Severin S. E. activate phosphodiesterase. But

due to the low activity of phosphodiesterase in healthy newborns, a more reasonable

explanation may be the assumption of a Са2+

-activating phosphodiesterase

mechanism [8] involving the phosphatidylinositol pathway [71]. This is confirmed by

our data on the decrease in the brush border of the epithelium of the small intestine in

patients with enteropathology of calfs containing phosphatidylinositol 2.4 times [4].

1.3.3 The intracellular content of the prostaglandins F1α and E2

In the gastrointestinal tract there are mainly prostaglandins of group E and F

[53, 54]. It is generally accepted that group E prostaglandins stimulate active

secretion of electrolytes in the intestine by stimulating adenylate cyclase, i.e., by

increasing the concentration of cAMP, whereas the prostaglandins F1α, in all

likelihood, along with the secretory effect, stimulate the motility of the small

intestine.

The results on the content of prostaglandins E2 and F1α in isolated small

intestine epithelium of bovine animals correspond to their biological level in tissues

[44, 48]. In healthy newborn calves, the level of F1 is 3 times lower and 5 times

lower than E2 in comparison with adult animals. In diabetic patients calves increase

in 4.1 times the content and in 6.3 times – E2 in comparison with healthy newborn

calves. Properties of prostaglandins to cause diarrhea are indicated by their parenteral

use [48], oral [27] or intestinal perfusion [46]. At the same time, if the biological

effect of exogenous prostaglandins is well understood, the functional significance of

endogenous prostaglandins in the small intestine is still not clear. The involvement of

38

prostaglandins in the inflammatory processes [10] including inflammatory processes

of the small intestine [46] made it possible to put forward the concept that in

inflammatory bowel diseases there is an uncontrolled increase in the level of

prostaglandins. In the opinion of [46], the high level of prostaglandins in diarrhea not

of coins is the root cause of the disease. In favor of this concept are data on the level

of prostaglandins and cAMP in isolated epithelium of the small intestine of patients

with diarrhea of calves. With a high content of prostaglandin E2, which is an

activator of adenylate cyclase, we observe the reverse, low cAMP. Therefore, the

ability of prostaglandins to cause diarrhea can be considered as a convenient model

for stimulating diarrhea, whereas their biological effect may be more versatile and

requires detailed study. Thus, the ability of prostaglandins of group E2 and F1α to

inhibit the activity of intestinal Nа+, К

+ -ATPase [27, 54], stimulate the secretion of

НСO3- [14] and chlorine [54].

Our preliminary studies showed a 5.3-fold decrease in the activity of the Nа+,

К+- ATP-ase of basolateral epithelial membranes [17, 39, 68–70], the change in the

acid-base balance of crocs towards acidosis [14–16], a decrease in the level of

chlorine in the blood of patients with diarrhea in newborn calves.

Therefore, along with the data obtained on the participation of cyclic guanosine

monophosphate in stimulating secretion in the small intestine of diabetic patients of

newborn calves, it is possible to assume that this pathology of prostaglandins E2 and

F1α, namely, their involvement in anion secretion, is also involved (Fig. 1.3)

39

Fig. 1.3. Hypothetical mechanism of the secretion of electrolytes and Н2О in

the epithelium of the small intestine, including the participation of cGMP and

prostaglandins, with enteropathology with diarrheal syndrome in newborn calves

40

CONCLUSION TO CHAPTER I

The modern concept of the development of enteropathology with diarrheal

syndrome (infectious and noninfectious nature) in the small intestine of animals and

humans provides for the direct participation of cyclic nucleotides and prostaglandins

in the initiation of secretory processes and inhibition of absorption.

Enteropathology of newborn calves is widespread, causing significant damage

to livestock and represents a great economic and social problem. Uncertainty of the

etiologic factor on the one hand, and a variety of causes that can lead to pathology

(content, feeding, quality and quantity of colostrum, its temperature, etc.), on the

other hand. Let us assume that the basis for the onset and development of diarrhea in

newborn calves may be a single biochemical "trigger" mechanism of Н20 secretion

and electrolytes localized in the epithelium of the small intestine. Obviously, for its

investigation it is necessary to use a sufficiently pure experimental material-isolated

epithelial cells, the procedure for obtaining which for cattle is not currently described.

In addition, given the high sensitivity of individual tissues of the body to respond to

fluctuations in cyclic nucleotides and prostaglandins on external influences (stresses,

etc.), as well as the specificity of the object of our research, we faced diverse

methodological problems in order to exclude the effect of inadequate factors and the

manifestation of artifacts.

Using a variety of methodological approaches – chemical (citrate/EDTA) and

enzymatic (collagenase, hyaduronidase and acryzyme III), we conducted a study on

the selection of optimal experimental conditions for the purpose of obtaining viable

isolated epithelium cells of the small intestine of cattle.

Criteria for the effectiveness of the method used were: a) the ability to

maximize the release of cells in the incubation medium (histological control, the

amount of cell protein); b) the homogeneity of the cell suspension, the absence of

aggregates of cell clusters (light microscopy); c) preservation of morphological

parameters (electron microscopy); d) cell nativity (Trypan blue staining, release into

41

the incubation medium of cytosolic lactate dehydrogenase); e) satisfactory values of

intravital metabolic parameters (state of glycolysis, etc.).

As shown by the studies, the most optimal criterion is the use of 0.05%

collagenase (enzymatic method), as well as the method using citrate/EDTA (chemical

method). Moreover, with satisfactory parameters, citrate/EDTA method is

economical.

But to study the level of cyclic nucleotides and prostaglandins this procedure is

not acceptable in view of the possibility of changing the concentration of calcium.

Thus, in the present work, two procedures for the production of epithelial cells

of the small intestine of adult cattle, newborn calves – healthy and patients with

enteropathology with diarrhea, were developed and proposed, and their metabolic

activity was assessed. The peculiarities of metabolic processes (a decreased state of

glycolysis, a cycle of tricarboxylic acids) in isolated small intestine epithelium of

healthy newborn calves are established, in comparison with adult animals, which can

characterize a special (colostrum) feeding period. In patients with enteropathology

with diarrhea syndrome of newborn calves, activation of glycolytic processes is

observed with simultaneous inhibition of oxidative processes, which, apparently,

reflects the pathological state of the tissue.

When studying the state of cyclic nucleotide exchange, we studied the content

of cAMP, cGMP and enzymes of their metabolism (adenylate cyclase, guanylate-

clase, phosphodiesterase) in freshly isolated isolated epithelial cells, as well as cAMP

and cGMP levels in blood and urine.

For adult cattle, it has been established (epithelium/blood/urine): cAMP –

59.4±10.9/34.5±2.1/18.2±4.8 pmol/g protein (or ml of liquid); cGMP - traces/tracks/

10.3±2.1 nmol/ml. Thus, the obtained value of the level of cAMP in the epithelium of

the small intestine of bovines characterizes its intravital level in the epithelial tissue

of this animal. The high level of cGMP in the urine reflects, apparently, the high

diuretic activity of the kidneys, due to the high water content in the feed.

42

In the physiological limits, there is also the activity of adenylate cyclase,

guanylate cyclase and phosphodiesterase of cyclic nucleotides in the epithelium of

adult cattle.

The content of prostaglandin E2 and F1α in isolated small intestine epithelium

of adult cattle is 168±36 and 205±55 ng/mg, respectively, which, apparently, reflects

their physiological level in the tissue.

For healthy newborn calves, the level of cAMP, cGMP and adenylate cyclase

activity is the same as for adult animals. The content of prostaglandins is significantly

lower, F1α is 3 times, E2 is 5-fold, which may reflect the peculiarities of the state of

the epithelium of the small intestine of newborn calves, as described for other animal

species.

Acute digestive disorders of newborn calves are accompanied by significant

changes in the studied indicators. Thus, in isolated epithelium of the small intestine of

diabetic patients of newborn calves, the level of cAMP decreases by 5.7 times, but

the level of cGMP rises tenfold. At the same time, a decrease in the activity of

adenylate cyclase, but an increase in the activity of guanylate cyclase (by 2.2 times),

and phosphodiesterase (possibly with the GMR of the stimulated isoform) has been

established. The obtained data allow us to conclude that the emergence and

development of enteropathology with diarrheal syndrome of alimentary nature in

newborn calves takes place with the participation of a biochemical pathway involving

cGMP-mediated secretion of H20 and electrolytes.

The high level in the epithelium of sick newborn animals of the prostaglandin

F1α (4.1 times) and E2 (6.3 times) may indicate a profound pathological changes that

are accompanied by damage to membranes, enhanced secretion of НС03- and Сl

-,

inflammatory phenomena. Indeed, based on recent data, an increase in the level of

prostaglandins in the pathology of the small intestine is a secondary process, but

capable of stimulating additional secretion of anions. This allows us to explain the

violation of the electrolyte balance that we received earlier in the body of sick

newborn calves, in particular – the development of the acidosis state, a sharp decrease

in С1- in the blood plasma.

43

Thus, as a result of the studies, the state of exchange of cyclic nucleotides and

prostaglandins in epithelium of the small intestine of cattle as an animal species was

characterized for the first time, the features of these biochemical regulatory systems

for the neonatal period were established. Based on the known role of cAMP, cGMP

and prostaglandins in the processes of secretion and absorption, the mechanisms of

the development of enteropathology of infectious and noninfectious nature, and

convincing data on the significantly high content of cGMP and prostaglandins in the

epithelium of the small intestine, the pathology suggests a possible biochemical

mechanism of diarrhea development in newborn calves, which included cGMP-

mediated secretion of fluid and electrolytes:

- unidentified etiologic factor (E. coli, rotavirus, Са2+

, etc.) lead to activation of

guanylate cyclase and increase in cGMP level;

- an increase in the intracellular concentration of cGMP leads to the activation

of secretory processes, as a result of which the secretion of Na+ predominates over its

absorption;

- H2O is secreted in parallel with sodium in its hy